It is well-known to transfer cloned genes into host cells as a means for producing the proteins expressed by such genes. Numerous eukaryotic host/vector systems have been used to express cloned genes. In general, the goal of such systems is to amplify the copies of the cloned gene and thus increase the yield of the protein expressed by such gene.
One known system uses recombinant bacterial plasmids that can be propagated and expressed in mammalian cells that carry the origin of DNA replication and transcriptional regulatory elements derived from the genome of simian virus (SV-40). See R. C. Mulligan and P. Berg, Science, 209:14-22 (1980); S. Subramami, R. C. Mulligan, P. Berg, Mol. Cell. Biol. 1, 854 (1981), P. J. Southern and P. Berg, 1:327 (1982); C. C. Simonson and A. D. Levinson; Proc. Nat'l Acad. Sci. USA, 80:2495 (1983). These vectors include the SV-40 T antigen gene. The expression of this gene is required to activate replication of the SV-40 origin in monkey cells.
Subsequently, it was discovered that the usefulness of these SV-40-derived vectors could be improved by using them with permissive cells (i.e. cells supporting the replication of the virus) carrying integrated copies of the T antigen (Y. Gluzman Cell 23, 175 (1980)). The permissive cells, termed COS (CV1-origin SV-40), are able to support replication of any DNA that contains an intact SV-40 origin sequence. These COS cells express the early viral gene product, T antigen, and are able to support replication of any DNA that contains an intact SV-40 origin sequence. The drawback of this system is that the COS cell line constitutively expressed active T antigen. Thus, it was not possible to regulate replication of SV40 vectors.
Regulation of gene expression is desirable for several reasons. For example, if a transfected gene product is toxic to the host, it would be advantageous to regulate (and particularly to defer) expression of that product.
In addition, extrachromosomal gene replication can in itself be lethal to the host cell. Thus, it is advantageous to be able to defer the onset of replication until a critical mass of host cells can be prepared for efficient production of the desired protein.
In unregulated systems, it is difficult to select for stable transformants since large amounts of extrachromosomal DNA are continuously present. Also, since the clones expressing the gene of interest may die quickly because they carry on extrachromosomal replication, it is possible that such clones may be overgrown by clones that have lost the sequences of interest.
The present invention involves extrachromosomal gene amplification mediated by a temperature-sensitive large T antigen provided in trans in permissive mouse cells and takes advantage of the finding of that the polyoma late promoter can direct the expression of foreign coding sequences linked to such promoter.
The present invention involves a system for amplification and expression of transfected genes by temperature induction. The host cells comprise permissive murine cells that contain (and can express) a polyoma virus large T antigen gene whose activity can be regulated by temperature shift. The cells permit the expression of functional T antigen at a second temperature and restrict the function of this antigen at a first (higher) temperature. When cultured at the lower (permissive) temperature the cells produce a functional large T antigen. The functional large T antigen acts on a functional virus origin of replication also present in the host cell and causes replication and excision of an integrated plasmid. As a result, the number of copies of plasmid DNA present in the cell is increased on a logarithmic basis. The level of expression of the desired gene product is also logarithmically increased.
In order to express large quantities of large T antigen, the host cell is transformed to contain an integrated polyoma large T gene with a non-functional (or mutated) origin of replication to shut off replication of viral DNA and autoregulation of the large T gene.
The transformed cell is transfected with a vector containing a functional origin (of the same virus species as the large T antigen) the gene coding for the protein of interest (with proper control signals), and a dominant selectable marker.
In fact, transfection is not required. A vector can be inserted by microinjection or by infection of the host cell with a retroviral vector.
Selectable markers include neomycin resistance gene, guanine phosphoribosyl transferase and others.
The large T antigen produced in the resulting cell by virtue of the first transformation is available to act on the vector to cause replication of the vector DNA, including the gene for the protein of interest. This is why it is necessary for the vector to contain a functional origin of replication of the same viral species as the large T.